During the processes of thymic lymphocyte maturation the thymus receives progenitors from hematopoietic tissues, regulates their rearrangement of different classes of T cell receptor genes, positively selects those whose receptors interact with self-MHC, ignores those who receptors do not, and actively deletes those that are potentially autoimmune. The stromal cells of the thymus that regulate this process are derived from all 3 tissue layers - endoderm, ectoderm, and mesoderm. During fetal life the thymus produces a class of fetal T cells that are exported to skin and reproductive epithelium, where they self-renew for life. Thereafter all T cells are produced throughout the life of the animal to protect against intracellular infections and newly developing cancers. In this grant several stages of thymic lymphocyte development are studied. Taking advantage of our recent isolation of several hematopoietic cells capable of giving rise to thymic lymphocytes (bone marrow long-term hematopoietic stem cells [LT-HSC]), ST-HSC, and non-self-renewing multipotent progenitors (MPP); the common lymphocyte progenitor (CLP), and the bone marrow common T cell progenitor (CTP), the identification of which of these progenitors emigrates to the thymus to initiate T cell development will be determined. Within the thymus, at least 2 pathways and up to 10 cells placed in lineages of these 2 pathways have been isolated and purified to homogeneity. In addition, the major subclasses of stromal cells have been identified, are capable of isolation, and are known to be useful in in vitro models of thymic lymphocyte differentiation. A major goal of this grant is to identify particular interactions between single stage developing T cells and single type stromal cells, including stroma that are involved in the processes of thymic maturation. These processes include 1) induction of rearrangement of the TCRgammadelta genes; 2) the TCRalphabeta genes; 3) finding the stroma responsible for positive selection of self MHC reactive T cells; 4) the stroma that house ignored cells that are destined to die by default; and 5) the stromal elements that actively delete or inactivate the small subset of self reactive T cells that would be autoimmune. Two model systems for the development of T lymphocytes predominate in the literature - normal lymphocytes responding to self superantigens (SAg), and developing T cells expressing transgenic TCRs against particular MHC, peptide antigens. The 2 models give markedly different answers as to the stages at which positive and negative selection occur. A major part of this grant is to develop a more physiological, in situ knockin of a particular TCR into the TCR Vbeta region, so that the stages of thymic lymphocyte maturation can be determined without the artifacts of superantigens and enforced expression of high levels of TCR. Finally, because adult thymuses cannot support the production of the fetal T cells that emigrate to skin and reproductive epithelium, and because adult HSC cannot respond to fetal thymic stroma by making fetal outcomes, several experiments will be carried out to identify and isolate the fetal stroma that are responsible for fetal T cell development and, if possible, the fetal stem cell or lymphocyte progenitor receptors that enable them to recognize and respond to the fetal stroma.